Design and optimization of effector-activated ribozyme ligases

A selected ribozyme ligase, L1, has been engineered to respond to small org anic effecters. Residues important for ribozyme catalysis were mapped to a compact core structure. Aptamers that bound adenosine and theophylline were appended to the core structure, and the resultant aptazymes proved to be r esponsive to their cognate effecters, Rational sequence substitutions in th e joining region between the aptamer and the ribozyme yielded aptazymes who se activities were enhanced from 800-1600-fold in the presence of 1 mM ATP or theophylline, respectively, However, when an anti-flavin aptamer was app ended to the core ribozyme structure flavin-responsivity was minimal, The j oining region between the aptamer and the ribozyme core was randomized and a series of negative and positive selection steps yielded aptazymes that we re activated by up to 260-fold in the presence of 100 mu M FMN, The selecte d joining regions proved to be 'communication modules' that could be used t o join other aptamers to the ribozyme core to form aptazymes. These results show that ribozyme ligases can be readily engineered to function as allost eric enzymes, and reveal that many of the techniques and principles previou sly demonstrated during the development of hammerhead aptazymes may be gene ralizable.